MSCA1 nucleotide sequences impacting plant male fertility and method of using same

ABSTRACT

Nucleotide sequences of a Msca1 gene, critical to male fertility in plants are described, with DNA molecule and amino acid sequences set forth. Promoter sequences and their essential regions are also identified. The nucleotide sequences are useful in impacting male fertility in plants.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of previously filed application U.S.Ser. No. 11/833,385, filed Aug. 3, 2007, now U.S. Pat. No. 7,915,478,the contents of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinating if pollenfrom one flower is transferred to the same or another flower of the sameplant or a genetically identical plant. A plant is cross-pollinated ifthe pollen comes from a flower on a different plant.

In certain species, such as Brassica campestris, the plant is normallyself-sterile and can only be cross-pollinated. In self-pollinatingspecies, such as soybeans and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower.

Maize plants (Zea mays L.) present a unique situation in that they canbe bred by both self-pollination and cross-pollination techniques. Maizehas male flowers, located on the tassel, and female flowers, located onthe ear, on the same plant. It can self or cross pollinate. Naturalpollination occurs in maize when wind blows pollen from the tassels tothe silks that protrude from the tops of the incipient ears.

The development of maize hybrids requires the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection are two of thebreeding methods used to develop inbred lines from populations. Breedingprograms combine desirable traits from two or more inbred lines orvarious broad-based sources into breeding pools from which new inbredlines are developed by selfing and selection of desired phenotypes. Ahybrid maize variety is the cross of two such inbred lines, each ofwhich may have one or more desirable characteristics lacked by the otheror which complement the other. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which have commercial potential. The hybrid progeny of thefirst generation is designated F₁. In the development of hybrids onlythe F₁ hybrid plants are sought. The F₁ hybrid is more vigorous than itsinbred parents. This hybrid vigor, or heterosis, can be manifested inmany ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the maletassel is removed from the growing female inbred parent, which can beplanted in various alternating row patterns with the male inbred parent.Consequently, providing that there is sufficient isolation from sourcesof foreign maize pollen, the ears of the female inbred will befertilized only with pollen from the male inbred. The resulting seed istherefore hybrid (F₁) and will form hybrid plants.

Field variation impacting plant development can result in plantstasseling after manual detasseling of the female parent is completed.Or, a female inbred plant tassel may not be completely removed duringthe detasseling process. In any event, the result is that the femaleplant will successfully shed pollen and some female plants will beself-pollinated. This will result in seed of the female inbred beingharvested along with the hybrid seed which is normally produced. Femaleinbred seed does not exhibit heterosis and therefore is not asproductive as F₁ seed. In addition, the presence of female inbred seedcan represent a germplasm security risk for the company producing thehybrid.

Alternatively, the female inbred can be mechanically detasseled bymachine. Mechanical detasseling is approximately as reliable as handdetasseling, but is faster and less costly. However, most detasselingmachines produce more damage to the plants than hand detasseling. Thus,no form of detasseling is presently entirely satisfactory, and a needcontinues to exist for alternatives which further reduce productioncosts and to eliminate self-pollination of the female parent in theproduction of hybrid seed.

A reliable system of genetic male sterility would provide advantages.The laborious detasseling process can be avoided in some genotypes byusing cytoplasmic male-sterile (CMS) inbreds. In the absence of afertility restorer gene, plants of a CMS inbred are male sterile as aresult of factors resulting from the cytoplasmic, as opposed to thenuclear, genome. Thus, this characteristic is inherited exclusivelythrough the female parent in maize plants, since only the femaleprovides cytoplasm to the fertilized seed. CMS plants are fertilizedwith pollen from another inbred that is not male-sterile. Pollen fromthe second inbred may or may not contribute genes that make the hybridplants male-fertile. Usually seed from detasseled normal maize and CMSproduced seed of the same hybrid must be blended to insure that adequatepollen loads are available for fertilization when the hybrid plants aregrown and to insure cytoplasmic diversity.

One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465and 4,727,219 to Brar, et al. However, this form of genetic malesterility requires maintenance of multiple mutant genes at separatelocations within the genome and requires a complex marker system totrack the genes and make use of the system convenient. Patterson alsodescribed a genic system of chromosomal translocations which can beeffective, but which are complicated. (See, U.S. Pat. Nos. 3,861,709 and3,710,511.)

Many other attempts have been made to improve on these systems. Forexample, Fabijanski, et al., developed several methods of causing malesterility in plants (see EPO 89/3010153.8 publication no. 329,308 andPCT application PCT/CA90/00037 published as WO 90/08828). One methodincludes delivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter. Another involves anantisense system in which a gene critical to fertility is identified andan antisense to the gene inserted in the plant. Fabijanski, et al. alsoshows several cytotoxic antisense systems. See EP0329308. Still othersystems use “repressor” genes which inhibit the expression of anothergene critical to male sterility. See PCT/GB90/00102, published as WO90/08829. For yet another example see U.S. Pat. No. 6,281,348.

A still further improvement of this system is one described at U.S. Pat.No. 5,478,369 in which a method of imparting controllable male sterilityis achieved by inactivating or otherwise silencing a gene native to theplant that is critical for male fertility and transforming that plantwith the gene critical to male fertility linked to an inducible promotercontrolling expression of the gene. That is, the expression of theendogenous sequence is prevented, by any of the methods known to askilled person in the art for preventing expression of a sequence (suchan antisense methods, cosuppression, mutation, use of ribozymes orhairpins, various repression systems and the like, discussed infra.) Theplant is thus constitutively sterile, becoming fertile only when thepromoter is induced and its linked male fertility gene is expressed.

In a number of circumstances, a male sterility plant trait is expressedby maintenance of a homozygous recessive condition. Difficulties arisein maintaining the homozygous condition, when a restoration gene must beused for maintenance. For example, a natural mutation in a gene criticalto male fertility can impart a male sterility phenotype to plants whenthis mutant allele is in the homozygous state. But because thishomozygosity results in male sterility, the homozygous male-sterile linecannot be maintained. Fertility is restored when the non-mutant form ofthe gene is introduced into the plant. However, this form of linemaintenance removes the desired homozygous recessive condition, restoresfull male fertility in half of the resulting progeny, and preventsmaintenance of pure male sterile maternal lines. These issues can beavoided where production of pollen containing the restoration gene iseliminated, thus providing a maintainer plant producing only pollen notcontaining the restoration gene, and the progeny retain their homozygouscondition when fertilized by such pollen. An example of one approach isshown in Dellaporta et al., U.S. Pat. No. 6,743,968, in which a plant isproduced having a hemizygotic construct comprising a gene that producesa product fatal to a cell, linked with a pollen-specific promoter, andthe restoration gene. When crossed with the homozygous recessive malesterile plant, the progeny thus retains the homozygous recessivecondition.

As noted, an essential aspect of much of the work underway with malesterility systems is the identification of genes impacting malefertility. Such a gene can be used in a variety of systems to controlmale fertility including those described herein.

Genetic male sterility results from a mutation, suppression, or otherimpact to one of the genes critical to a specific step inmicrosporogenesis, the term applied to the entire process of pollenformation. These genes can be collectively referred to as male fertilitygenes (or, alternatively, male sterility genes). There are many steps inthe overall pathway where gene function impacts fertility. This seemsaptly supported by the frequency of genetic male sterility in maize. Newalleles of male sterility mutants are uncovered in materials that rangefrom elite inbreds to unadapted populations.

At U.S. Pat. No. 5,478,369 there is described a method by which the Ms45male fertility gene was tagged and cloned on maize chromosome 9.Previously, there had been described a male sterility gene on chromosome9, ms2, which had never been cloned and sequenced. It is not allelic tothe gene referred to in the '369 patent. See Albertsen, M. and Phillips,R. L., “Developmental Cytology of 13 Genetic Male Sterile Loci in Maize”Canadian Journal of Genetics & Cytology 23:195-208 (January 1981). Theonly fertility gene cloned before that had been the Arabadopsis genedescribed at Aarts, et al., supra.

Examples of genes that have been discovered subsequently that arecritical to male fertility are numerous and include the ArabidopsisABORTED MICROSPORES (AMS) gene, Sorensen et al., The Plant Journal(2003) 33(2):413-423); the Arabidopsis MS1 gene (Wilson et al., ThePlant Journal (2001) 39(2):170-181); the NEF1 gene (Ariizumi et al., ThePlant Journal (2004) 39(2):170-181); Arabidopsis AtGPAT1 gene (Zheng etal., The Plant Cell (2003) 15:1872-1887); the Arabdiopsis dde2-2mutation was shown to be defective in the allene oxide syntase gene(Malek et al., Planta (2002)216:187-192); the Arabidopsis facelesspollen-1 gene (flp1) (Ariizumi et al, Plant Mol. Biol. (2003)53:107-116); the Arabidopisis MALE MEIOCYTE DEATH1 gene (Yang et al.,The Plant Cell (2003) 15: 1281-1295); the tapetum-specific zinc fingergene, TAZ1 (Kapoor et al., The Plant Cell (2002) 14:2353-2367); and theTAPETUM DETERMINANT1 gene (Lan et al, The Plant Cell (2003)15:2792-2804).

The table below lists a number of known male fertility mutants or genesfrom Zea mays.

GENE NAME ALTERNATE NAME REFERENCE ms1 male sterile1 male sterile1, ms1Singleton, WR and Jones, DF. 1930. J Hered 21: 266-268 ms10 malesterile10 male sterile10, ms10 Beadle, GW. 1932. Genetics 17: 413-431ms11 male sterile11 ms11, male sterile11 Beadle, GW. 1932. Genetics 17:413-431 ms12 male sterile12 ms12, male sterile12 Beadle, GW. 1932.Genetics 17: 413-431 ms13 male sterile13 ms*-6060, male sterile13, ms13Beadle, GW. 1932. Genetics 17: 413-431 ms14 male sterile14 ms14, malesterile14 Beadle, GW. 1932. Genetics 17: 413-431 ms17 male sterile17ms17, male sterile17 Emerson, RA. 1932. Science 75: 566 ms2 malesterile2 male sterile2, ms2 Eyster, WH. 1931. J Hered 22: 99-102 ms20male sterile20 ms20, male sterile20 Eyster, WH. 1934. Genetics of Zeamays. Bibliographia Genetica 11: 187-392 ms23 male sterile23 ms*-6059,ms*-6031, ms*- West, DP and Albertsen, MC. 6027, ms*-6018, ms*-6011,1985. MNL 59: 87 ms35, male sterile23, ms*- Bear7, ms23 ms24 malesterile24 ms24, male sterile24 West, DP and Albertsen, MC. 1985. MNL 59:87 ms25 male sterile25 ms*-6065, ms*-6057, Loukides, CA; Broadwater, AH;ms25, male sterile25, ms*- Bedinger, PA. 1995. 6022 Am J Bot 82:1017-1023 ms27 male sterile27 ms27, male sterile27 Albertsen, MC. 1996.MNL 70: 30-31 ms28 male sterile28 ms28, male sterile28 Golubovskaya, IN.1979. MNL 53: 66-70 ms29 male sterile29 male sterile29, ms*-JH84A,Trimnell, MR et al. 1998. ms29 MNL 72: 37-38 ms3 male sterile3 Group 3,ms3, male sterile3 Eyster, WH. 1931. J Hered 22: 99-102 ms30 malesterile30 ms30, msx, ms*-6028, ms*- Albertsen, MC et al. 1999. Li89,male sterile30, ms*- MNL 73: 48 LI89 ms31 male sterile31 ms*-CG889D,ms31, male Trimnell, MR et al. 1998. sterile31 MNL 72: 38 ms32 malesterile32 male sterile32, ms32 Trimnell, MR et al. 1999. MNL 73: 48-49ms33 male sterile33 ms*-6054, ms*-6024, Patterson, EB. 1995. MNL ms33,ms*-GC89A, ms*- 69: 126-128 6029, male sterile6019, Group 7, ms*-6038,ms*- Stan1, ms*-6041, ms*- 6019, male sterile33 ms34 male sterile34Group 1, ms*-6014, ms*- Patterson, EB. 1995. MNL 6010, male sterile34,ms34, 69: 126-128 ms*-6013, ms*-6004, male sterile6004 ms36 malesterile36 male sterile36, ms*-MS85A, Trimnell, MR et al. 1999. ms36 MNL73: 49-50 ms37 male sterile 37 ms*-SB177, ms37, male Trimnell, MR et al.1999. sterile 37 MNL 73: 48 ms38 male sterile38 ms30, ms38, ms*-WL87A,Albertsen, MC et al. 1996. male sterile38 MNL 70: 30 ms43 male sterile43ms43, male sterile43, ms29 Golubovskaya, IN. 1979. Int Rev Cytol 58:247-290 ms45 male sterile45 Group 6, male sterile45, Albertsen, MC; Fox,TW; ms*-6006, ms*-6040, ms*- Trimnell, MR. 1993. Proc BS1, ms*-BS2,ms*-BS3, Annu Corn Sorghum Ind ms45, ms45′-9301 Res Conf 48: 224-233ms48 male sterile48 male sterile48, ms*-6049, Trimnell, M et al. 2002.ms48 MNL 76: 38 ms5 male sterile5 ms*-6061, ms*-6048, ms*- Beadle, GW.1932. 6062, male sterile5, ms5 Genetics 17: 413-431 ms50 male sterile50ms50, male sterile50, ms*- Trimnell, M et al. 2002. 6055, ms*-6026 MNL76: 39 ms7 male sterile7 ms7, male sterile7 Beadle, GW. 1932. Genetics17: 413-431 ms8 male sterile8 male sterile8, ms8 Beadle, GW. 1932.Genetics 17: 413-431 ms9 male sterile9 Group 5, male sterile9, ms9Beadle, GW. 1932. Genetics 17: 413-431 ms49 male sterile49 ms*-MB92,ms49, male Trimnell, M et al. 2002. sterile49 MNL 76: 38-39

There remains a need to identify nucleotide sequences critical to malefertility in plants. There also remains a need to identify regulatoryregions which preferentially direct expression to male tissue of aplant.

In the present invention the inventors provide novel DNA molecules andthe amino acid sequence encoded that are critical to male fertility inplants. These can be used in any of the systems where control offertility is useful, including those described above.

Thus, one object of the invention is to provide a nucleic acid sequence,the expression of which is critical to male fertility in plants and inwhich a mutation of the sequence causes male sterility when in thehomozygous state.

Another object is to provide regulatory regions that preferentiallydirect expression of operably linked nucleotide sequences to maletissue(s) of a plant.

A further object of the invention is to provide a method of using suchnucleotide sequences to mediate male fertility in plants.

Further objects of the invention will become apparent in the descriptionand claims that follow.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid sequences, and, specifically, DNAmolecules and the amino acid encoded by the DNA molecules, which arecritical to male fertility. Impacting the functional expression of suchsequences results in the mediation of male fertility. Regulatory regionsdirecting expression preferentially to male tissue are also provided.The invention also relates to use of such nucleotide sequences tomediate fertility in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a locus map of the male fertility gene Msca1.

FIG. 2 is the nucleotide sequence of the Msca1 gene (SEQ ID NO: 1)

FIG. 3 is the protein sequence of Msca1 (SEQ ID NO: 2)

FIG. 4 is the msca1-ref nucleotide sequence (SEQ ID NO: 3)

FIG. 5 is an alignment of fertile and sterile msca1-mg12 alleles, (thenucleotide sequence of the fertile is SEQ ID NO: 4; the protein sequenceis SEQ ID NO: 5; the nucleotide sequence of msca1-mg12 sterile is SEQ IDNO: 6 and the protein sequence is SEQ ID NO: 7). The fertile allelesequence contains an additional 490 base pairs deleted from the 3′region of the sterile sequence.

FIG. 6 shows alignment of the Msca1 wildtype gene from the corn hybridMissouri 17 (Mo17) (SEQ ID NO: 8) with msca1-mg12 alleles in a fertileplant (Mg12-Fert) (SEQ ID NO: 9) and a sterile plant (Mg12-Ster) (SEQ IDNO:10). The circled region refers to the CCMC redox motif (SEQ ID NO:11) and the gluteredoxin binding site (GSH Binding) (SEQ ID NO: 12) isunderlined.

FIG. 7A-7F shows alignment of the msca1 alleles, Ms22-6036 from afertile plant (Fert) SEQ ID NO: 13, coding SEQ ID NO: 23) with a sterileplant (6036s) (SEQ ID NO: 14, coding SEQ ID NO: 24). The sterilesequence contains an 850 base pair insertion at the 3′ end. Theinsertion contains small perfect TIRs of eight basepairs (indicated at“TIR”) with about 200 basepairs of a transposon-like sequence.

FIG. 8 shows a graphic alignment of the Msca1 sequence with mutantalleles msca1-ref, msca1-mg12 and msca1-6036.

FIG. 9 is the full length promoter of Msca1 (SEQ ID NO: 15)

FIG. 10 is the nucleotide sequence of the rice Msca1 gene (SEQ ID NO:16)

FIG. 11 is the protein sequence of rice Msca1 (SEQ ID NO: 17)

FIG. 12 is the full length promoter of rice Msca1 (SEQ ID NO: 18)

FIG. 13 is the sequence of the rice msca1 allele (SEQ ID NO: 19).

DISCLOSURE OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

The invention includes using the sequences shown herein to impact malefertility in a plant, that is, to control male fertility by manipulationof the genome using the genes of the invention. By way of example,without limitation, any of the methods described infra can be used withthe sequence of the invention such as introducing a mutant sequence intoa plant to cause sterility, causing mutation to the native sequence,introducing an antisense of the sequence into the plant, use of hairpinformations, linking it with other sequences to control its expression,or any one of a myriad of processes available to one skilled in the artto impact male fertility in a plant.

The Msca1 gene (also referred to as Ms22) described herein is located onshort arm of maize chromosome 7 and its dominant allele encodes aprotein critical to male fertility. The locus map is represented atFIG. 1. The Msca1 gene can be used in the systems described above, andother systems impacting male fertility.

Mutations referred to as ms22 or msca1 were first noted asphenotypically male sterile with anthers did not exude from the tasseland lacked sporogenous tissue. West and Albertsen (1985) MaizeNewsletter 59:87; Neuffer et al. (1977) Mutants of maize. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. The mutant locus wasoriginally referred to as ms22 but was later changed to msca1, or malesterile converted anther. See Chaubal et al. “The transformation ofanthers in the msca1 mutant of maize” Planta (2003)216:778-788.

Study of the mutant included collecting anthers from young spikelets ofimmature tassels in plant families segregating 1:1 for male sterile/malefertility plants for microscopic study. Using an F₂ family segregatingfor the msca1 mutation, DNA was isolated from male sterile plants,electrophoresed and hybridized with restriction fragment lengthpolymorphism markers, and mapped to chromosome 7. See Chaubal et al.“The transformation of anthers in the msca1 mutant of maize” Planta(2003)216:778-788.

The msca1 mutants are unusual in that stamen primordia develop normally,but differentiation and cell division do not occur, with the tissueinstead developing into nonfunctional vascular tissue. There is noasymmetric division of archesporial cells into large primary sporogenousand smaller primary parietal cells. Instead, the anther containsparenchymal cells and non-functional vascular strands with no formationof normal anther cells such as microspores, tapetum, middle layer andendothecium. All of the cell layers of the anther convert in mutantplants into vegetative structures. Since the Msca1 gene operates afterstamen primordial initiation and before division of the archesporialcells, interruption of gene expression acts as a developmental block. Asopposed to other male sterility genes such as MAC1, EMS1 or GNE2(Sorensen et al. (2002) Plant J. 29:581-594) rather than breaking downcells in the quartet stage, microspores never develop. Mutations in theSPOROCYTELESS/NOZZLE gene act early in development, but impact bothanther and ovule formation such that plants are male and female sterile.Yang et al. The SPOROCYTELESS gene of Arabidopsis is required forinitiation of sporogenesis and encodes a novel nuclear protein.GenesDev. 1999 Aug. 15; 13(16):2108-17. The Msca1 gene expression wheninterrupted does not impact floral tissue. Rather, the anther istransformed into a vegetative structure and microsporogenesis neverbegins and the end result is greatly increased reliability inmaintenance of male sterility.

The invention is also directed to impacting male fertility of a plant byimpacting the Msca1 nucleotide sequence. Impacting male fertility refersto a change in the male fertility of the plant from the fertilityphenotype prior to impacting the nucleotide sequence. It may result inmale sterility, as when the sequence is impacted such that expression ofthe Msca1 male fertility critical gene does not occur as in thewild-type condition. The fertility of a plant may also be impacted by,for example, introducing into a plant that comprises a mutated msca1allele, a Msca1 nucleotide sequence which restores fertility. Clearly,many variations are possible in impacting male fertility depending uponthe specific application. Impacting the Msca1 nucleotide sequence can beaccomplished using many tools available to one skilled in the art, asdiscussed in examples below. By way of example, the gene may contain aninsertion, such as that shown in msca1-6036 allele, or have a deletion,such as with msca1-mg12 allele. Use of mutagenesis, antisense genes,co-suppression, hairpin formations, selecting for mutant plants,insertion of one or more additional sequences which act to disrupt thegene expression are a few examples of the many means available tointerrupt expression of the Msca1 gene. Further, the invention isdirected to restoring male fertility in a plant having expression ofMsca1 disrupted, by introducing into the plant the wild-type Msca1complementary sequence.

It will be evident to one skilled in the art that variations, mutations,derivations including fragments smaller than the entire sequence setforth may be used which retain the male sterility controlling propertiesof the gene. As used herein, a “functional fragment” of the Msca1sequence is a nucleotide sequence that is formed by one or moredeletions from the entire sequence and which retains the functional ofbeing critical for male fertility. One of ordinary skill in the art canreadily assess the variant or fragment by its introduction into plantshomozygous for a stable male sterile allele of Msca1, followed byobservation of the plant's male tissue development.

The sequences of the invention may be isolated from any plant,including, but not limited to corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), millet (Panicum spp.), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats (Avena sativa), barley (Hordeum vulgare), vegetables,ornamentals, and conifers. Preferably, plants include corn, soybean,sunflower, safflower, canola, wheat, barley, rye, alfalfa, rice, cottonand sorghum.

Sequences from other plants may be isolated according to well-knowntechniques based on their sequence homology to the homologous codingregion of the sequences set forth herein. In these techniques, all orpart of the known coding sequence is used as a probe which selectivelyhybridizes to other sequences present in a population of cloned genomicDNA fragments (i.e. genomic libraries) from a chosen organism. Methodsare readily available in the art for the hybridization of nucleic acidsequences. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

Thus the invention also includes those nucleotide sequences whichselectively hybridize to the Msca1 nucleotide sequences under stringentconditions. In referring to a sequence that “selectively hybridizes”with Msca1, the term includes reference to hybridization, understringent hybridization conditions, of a nucleic acid sequence to thespecified nucleic acid target sequence to a detectably greater degreethan its hybridization to non-target nucleic acid.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences. Stringent conditions are target-sequence-dependent and willdiffer depending on the structure of the polynucleotide. By controllingthe stringency of the hybridization and/or washing conditions, targetsequences can be identified which are 100% complementary to a probe(homologous probing). Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected (heterologous probing). Generally, probes ofthis type are in a range of about 1000 nucleotides in length to about250 nucleotides in length.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See also Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

In general, sequences that correspond to the nucleotide sequences of thepresent invention and hybridize to the nucleotide sequence disclosedherein will be at least 50% homologous, 70% homologous, and even 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%homologous or more with the disclosed sequence. That is, the sequencesimilarity between probe and target may range, sharing at least about50%, about 70%, and even about 85% or more sequence similarity.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, stringent wash temperature conditions areselected to be about 5° C. to about 2° C. lower than the melting point(Tm) for the specific sequence at a defined ionic strength and pH. Themelting point, or denaturation, of DNA occurs over a narrow temperaturerange and represents the disruption of the double helix into itscomplementary single strands. The process is described by thetemperature of the midpoint of transition, Tm, which is also called themelting temperature. Formulas are available in the art for thedetermination of melting temperatures.

Preferred hybridization conditions for the nucleotide sequence of theinvention include hybridization at 42° C. in 50% (w/v) formamide, 6×SSC,0.5% (w/v) SDS, 100 (g/ml salmon sperm DNA. Exemplary low stringencywashing conditions include hybridization at 42° C. in a solution of2×SSC, 0.5% (w/v) SDS for 30 minutes and repeating. Exemplary moderatestringency conditions include a wash in 2×SSC, 0.5% (w/v) SDS at 50° C.for 30 minutes and repeating. Exemplary high stringency conditionsinclude a wash in 0.1×SSC, 0.1% (w/v) SDS, at 65° C. for 30 minutes toone hour and repeating. Sequences that correspond to the promoter of thepresent invention may be obtained using all the above conditions. Forpurposes of defining the invention, the high stringency conditions areused.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, or 100nucleotides in length, or longer. Those of skill in the art understandthat to avoid a high similarity to a reference sequence due to inclusionof gaps in the polynucleotide sequence a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of aligning sequences for comparison are well-known in the art.Thus, the determination of percent sequence identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4: 11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the globalalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local-alignment-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153;Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al.(1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller(1988) supra. A PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. The BLAST programs of Altschul et al (1990) J.Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul(1990) supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3 and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2; and theBLOSUM62 scoring matrix or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizesthe number of matches and minimizes the number of gaps. GAP considersall possible alignments and gap positions and creates the alignment withthe largest number of matched bases and the fewest gaps. It allows forthe provision of a gap creation penalty and a gap extension penalty inunits of matched bases. GAP must make a profit of gap creation penaltynumber of matches for each gap it inserts. If a gap extension penaltygreater than zero is chosen,

GAP must, in addition, make a profit for each gap inserted of the lengthof the gap times the gap extension penalty. Default gap creation penaltyvalues and gap extension penalty values in Version 10 of the GCGWisconsin Genetics Software Package for protein sequences are 8 and 2,respectively. For nucleotide sequences the default gap creation penaltyis 50 while the default gap extension penalty is 3. The gap creation andgap extension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 200. Thus, for example, thegap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

Identity to the sequence of the present invention would mean apolynucleotide sequence having at least 65% sequence identity, morepreferably at least 70% sequence identity, more preferably at least 75%sequence identity, more preferably at least 80% identity, morepreferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity.

The promoter of the Msca1 gene is also the subject of the presentinvention, as shown in the 1132 base pair sequence of FIG. 9 (SEQ ID NO:15). The regulatory region of the gene comprises bases 1 to 1132 of FIG.9, SEQ ID NO: 15 and other functional fragments of same. Promoterregions can be readily identified by one skilled in the art. Theputative start codon containing the ATG motif is identified at base 1133of SEQ ID NO: 1 (See FIG. 2) and upstream from the start codon is thepresumptive promoter.

By “promoter” is intended a regulatory region of DNA usually comprisinga TATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular coding sequence. A promoter can additionally comprise otherrecognition sequences generally positioned upstream or 5′ to the TATAbox, referred to as upstream promoter elements, which influence thetranscription initiation rate. It is recognized that having identifiedthe nucleotide sequences for the promoter region disclosed herein, it iswithin the state of the art to isolate and identify further regulatoryelements in the region upstream of the TATA box from the particularpromoter region identified herein. Thus the promoter region disclosedherein is generally further defined by comprising upstream regulatoryelements such as those responsible for tissue and temporal expression ofthe coding sequence, enhancers and the like. In the same manner, thepromoter elements which enable expression in the desired tissue such asmale tissue can be identified, isolated, and used with other corepromoters to confirm male tissue-preferred expression. By core promoteris meant the minimal sequence required to initiate transcription, suchas the sequence called the TATA box which is common to promoters ingenes encoding proteins. Thus the upstream promoter of Msca1 canoptionally be used in conjunction with its own or core promoters fromother sources. The promoter may be native or non-native to the cell inwhich it is found.

By way of example, a putative TATA box can be identified by primerextension analysis as described in by Current Protocols in MolecularBiology, Ausubel, F. M. et al. eds; John Wiley and Sons, New York pp.4.8.1-4.8.5 (1987). Regulatory regions of anther genes, such aspromoters, may be identified in genomic subclones using functionalanalysis, usually verified by the observation of reporter geneexpression in anther tissue and a lower level or absence of reportergene expression in non-anther tissue. The possibility of the regulatoryregions residing “upstream” or 5′ ward of the translational start sitecan be tested by subcloning a DNA fragment that contains the upstreamregion into expression vectors for transient expression experiments. Itis expected that smaller subgenomic fragments may contain the regionsessential for male-tissue preferred expression. For example, theessential regions of the CaMV 19S and ³⁵S promoters have been identifiedin relatively small fragments derived from larger genomic pieces asdescribed in U.S. Pat. No. 5,352,605.

The selection of an appropriate expression vector with which to test forfunctional expression will depend upon the host and the method ofintroducing the expression vector into the host and such methods arewell known to one skilled in the art. For eukaryotes, the regions in thevector include regions that control initiation of transcription andcontrol processing. These regions are operably linked to a reporter genesuch as CYP, UidA, encoding glucuronidase (GUS), or luciferase asdescribed herein. Expression vectors containing putative regulatoryregions located in genomic fragments can be introduced into intacttissues such as staged anthers, embryos or into callus. Methods of DNAdelivery are described below. For the transient assay system, variousanalysis may be employed. In one example, staged, isolated anthers areimmediately placed onto tassel culture medium (Pareddy, D. R. and J. F.Petelino, Crop Sci. J.; Vol. 29; pp. 1564-1566; (1989)) solidified with0.5% Phytagel (Sigma, St. Louis) or other solidifying media. Theexpression vector DNA is introduced within 5 hours preferably bymicroprojectile-mediated delivery with 1.2 μm particles at 1000-1100Psi. After DNA delivery, the anthers are incubated at 26° C. upon thesame tassel culture medium for 17 hours and analyzed by preparing awhole tissue homogenate and assaying for GUS or for lucifierase activity(see Gruber, et al., supra).

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Less than the entire promoter region can beutilized and the ability to drive anther-preferred expression retained.However, it is recognized that expression levels of mRNA can bedecreased with deletions of portions of the promoter sequence. Thus, thepromoter can be modified to be a weak or strong promoter. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. Generally, atleast about 30 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence. It is recognized that toincrease transcription levels, enhancers can be utilized in combinationwith the promoter regions of the invention. Enhancers are nucleotidesequences that act to increase the expression of a promoter region.Enhancers are known in the art and include the SV40 enhancer region, the35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′UTR) Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon. The term “isolated” refers to material such as anucleic acid or protein which is substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in it naturally occurring environment, or if the material is inits natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in a cellother than the locus native to the material. Methods for isolation ofpromoter regions are well known in the art.

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” of the regulatory sequence is anucleotide sequence that is a regulatory sequence variant formed by oneor more deletions from a larger sequence. For example, the 5′ portion ofa promoter up to the TATA box near the transcription start site can bedeleted without abolishing promoter activity, as described byOpsahl-Sorteberg, H-G. et al., “Identification of a 49-bp fragment ofthe HvLTP2 promoter directing aleruone cell specific expression” Gene341:49-58 (2004). Such variants should retain promoter activity,particularly the ability to drive expression in male tissues. Activitycan be measured by Northern blot analysis, reporter activitymeasurements when using transcriptional fusions, and the like. See, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.),herein incorporated by reference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

Sequences which hybridize to the regulatory sequences of the presentinvention are within the scope of the invention. Sequences thatcorrespond to the promoter sequences of the present invention andhybridize to the promoter sequences disclosed herein will be at least50% homologous, 70% homologous, and even 85% 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous or more with thedisclosed sequence.

Smaller fragments may yet contain the regulatory properties of thepromoter so identified and deletion analysis is one method ofidentifying essential regions. Deletion analysis can occur from both the5′ and 3′ ends of the regulatory region. Fragments can be obtained bysite-directed mutagenesis, mutagenesis using the polymerase chainreaction and the like. (See, Directed Mutagenesis: A Practical ApproachIRL Press (1991)). The 3′ deletions can delineate the essential regionand identify the 3′ end so that this region may then be operably linkedto a core promoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the essentialregion plus a core promoter. By core promoter is meant the sequencecalled the TATA box which is common to promoters in all genes encodingproteins. Thus the upstream promoter of Msca1 can optionally be used inconjunction with its own or core promoters from other sources. Thepromoter may be native or non-native to the cell in which it is found.

The core promoter can be any one of known core promoters such as theCauliflower Mosaic Virus 35S or 19S promoter (U.S. Pat. No. 5,352,605),ubiquitin promoter (U.S. Pat. No. 5,510,474) the 1N2 core promoter (U.S.Pat. No. 5,364,780) or a Figwort Mosaic Virus promoter (Gruber, et al.“Vectors for Plant Transformation” Methods in Plant Molecular Biologyand Biotechnology) et al. eds, CRC Press pp. 89-119 (1993)).

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the promotersequence set forth herein. In these techniques, all or part of the knownpromoter sequence is used as a probe which selectively hybridizes toother sequences present in a population of cloned genomic DNA fragments(i.e. genomic libraries) from a chosen organism. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al., eds., (1990) PCR Protocols,A Guide to Methods and Applications, Academic Press).

Further, a promoter of the present invention can be linked withnucleotide sequences other than the Msca1 gene to express otherheterologous nucleotide sequences. The nucleotide sequence for thepromoter of the invention, as well as fragments and variants thereof,can be provided in expression cassettes along with heterologousnucleotide sequences for expression in the plant of interest, moreparticularly in the male tissue of the plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence to be under the transcriptional regulation ofthe promoter. These expression cassettes are useful in the geneticmanipulation of any plant to achieve a desired phenotypic response.

Examples of other nucleotide sequences which can be used as theexogenous gene of the expression vector with the Msca1 promoter, orother promoters taught herein or known to those of skill in the artinclude complementary nucleotidic units such as antisense molecules(callase antisense RNA, barnase antisense RNA and chalcone synthaseantisense RNA, Ms45 antisense RNA), ribozymes and external guidesequences, an aptamer or single stranded nucleotides. The exogenousnucleotide sequence can also encode carbohydrate degrading or modifyingenzymes, amylases, debranching enzymes and pectinases, such as the alphaamylase gene, auxins, rol B, cytotoxins, diptheria toxin, DAM methylase,avidin, or may be selected from a prokaryotic regulatory system. By wayof example, Mariani, et al., Nature Vol. 347; pp. 737; (1990), haveshown that expression in the tapetum of either Aspergillus oryzaeRNase-T1 or an RNase of Bacillus amyloliquefaciens, designated“barnase,” induced destruction of the tapetal cells, resulting in maleinfertility. Quaas, et al., Eur. J. Biochem. Vol. 173: pp. 617 (1988),describe the chemical synthesis of the RNase-T1, while the nucleotidesequence of the barnase gene is disclosed in Hartley, J. Molec. Biol.;Vol. 202: pp. 913 (1988). The rolB gene of Agrobacterium rhizogenescodes for an enzyme that interferes with auxin metabolism by catalyzingthe release of free indoles from indoxyl-β-glucosides. Estruch, et al.,EMBO J. Vol. 11: pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet.;Vol. 84: pp. 520 (1992), have shown that the anther-specific expressionof the rolB gene in tobacco resulted in plants having shriveled anthersin which pollen production was severely decreased and the rolB gene isan example of a gene that is useful for the control of pollenproduction. Slightom, et al., J. Biol. Chem. Vol. 261: pp. 108 (1985),disclose the nucleotide sequence of the rolB gene. DNA moleculesencoding the diphtheria toxin gene can be obtained from the AmericanType Culture Collection (Rockville, Md.), ATCC No. 39359 or ATCC No.67011 and see Fabijanski, et al., E.P. Appl. No. 90902754.2, “MolecularMethods of Hybrid Seed Production” for examples and methods of use. TheDAM methylase gene is used to cause sterility in the methods discussedat U.S. Pat. No. 5,689,049 and PCT/US95/15229 Cigan, A. M. andAlbertsen, M. C., “Reversible Nuclear Genetic System for Male Sterilityin Transgenic Plants.” Also see discussion of use of the avidin gene tocause sterility at U.S. Pat. No. 5,962,769 “Induction of Male Sterilityin Plants by Expression of High Levels of Avidin” by Albertsen et al.

The invention includes vectors with the Msca1 gene and/or its promoter.A vector is prepared comprising Msca1, a promoter that will driveexpression of the gene in the plant and a terminator region. As noted,the promoter in the construct may be the native promoter or asubstituted promoter which will provide expression in the plant. Thepromoter in the construct may be an inducible promoter, so thatexpression of the sense or antisense molecule in the construct can becontrolled by exposure to the inducer. In this regard, aplant-compatible promoter element can be employed in the construct,influenced by the end result desired. When linking the Msca1 nucleotidesequence with another promoter, it will be preferable that the promoterdrive expression of the sequence sufficiently early in plant developmentthat the Msca1 sequence or fragment or variant is expressed afterprimordial initiation but before division of archesporial cells.Examples of the variety of promoters that could be used include theconstitutive viral promoters such as the cauliflower mosaic virus (CaMV)19S and 35S promoters or the figwort mosaic virus 35S promoter. See Kayet al., (1987) Science 236:1299 and European patent application No. 0342 926; and the ubiquitin promoter (see for example U.S. Pat. No.5,510,474) or any other ubiquitin-like promoter, which encodes aubiquitin protein, but may have varying particular sequences (forexample U.S. Pat. Nos. 5,614,399 and 6,054,574).

It will be evident to one skilled in the art that the construct can alsocontain one of the variety of other promoters available, depending uponthe particular application. For example, the promoter may be linked witha selectable marker, or a gene of interest for expression in the plantcell. In this regard, any plant-compatible promoter can be employed.Those can be the 35S and ubiquitin-like promoters as referred to above,or any other plant gene promoters, such as, for example, the promoterfor the small subunit of ribulose-1,5-bis-phosphate carboxylase, orpromoters from the tumor-inducing plasmids from Agrobacteriumtumefaciens, such as the nopaline synthase and octopine synthasepromoters; the barley lipid transfer protein promoter, LTP2 (Kalla etal., Plant J. (1994) 6(6): 849-60); the END2 promoter (Linnestad et al.U.S. Pat. No. 6,903,205); and the polygalacturonase PG47 promoter (SeeAllen and Lonsdale, Plant J. (1993) 3:261-271; WO 94/01572; U.S. Pat.No. 5,412,085See international application WO 91/19806 for a review ofillustrative plant promoters suitably employed in the present invention.

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the action of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol. 22: 361-366 (1993). Exemplary inducible promotersinclude ecdysone receptor promoters, U.S. Pat. No. 6,504,082; promotersfrom the ACE1 system which responds to copper (Mett et al. PNAS 90:4567-4571 (1993)); In2-1 and In2-2 gene from maize which respond tobenzenesulfonamide herbicide safeners (U.S. Pat. No. 5,364,780; Hersheyet al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al., Mol.Gen. Genetics 243: 32-38 (1994)); the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides; and the tobacco PR-1a promoter, which isactivated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156).

Tissue-preferred promoters can be utilized to target enhancedtranscription and/or expression within a particular plant tissue.Promoters may express in the tissue of interest, along with expressionin other plant tissue, may express strongly in the tissue of interestand to a much lesser degree than other tissue, or may express highlypreferably in the tissue of interest. Tissue-preferred promoters includethose described in Yamamoto et al. (1997) Plant J. 12(2): 255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al.(1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535;Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto etal. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl.Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3): 495-505. Inone embodiment, the promoters are those which preferentially express tothe male or female tissue of the plant. The invention does not requirethat any particular male tissue-preferred promoter be used in theprocess, and any of the many such promoters known to one skilled in theart may be employed. The native Msca1 promoter described herein is oneexample of a useful promoter. Another such promoter is the 5126promoter, which preferentially directs expression of the gene to whichit is linked to male tissue of the plants, as described in U.S. Pat.Nos. 5,837,851 and 5,689,051. Other examples include the Ms45 promoterdescribed at U.S. Pat. No. 6,037,523; Ms26 promoter described at USPublication No. 20060015968; SF3 promoter described at U.S. Pat. No.6,452,069; the BS92-7 promoter described at WO 02/063021; a SGB6regulatory element described at U.S. Pat. No. 5,470,359; the TA29promoter (Koltunow et al. (1990) “Different temporal and spatial geneexpression patterns occur during anther development.” Plant Cell2:1201-1224; Goldberg, R. B., Beals, T. P. and Sanders, P. M., (1993)“Anther development: basic principles and practical applications” PlantCell 5:1217-1229; and U.S. Pat. No. 6,399,856); the type 2metallothionein-like gene promoter (Charbonnel-Campaa et al., Gene(2000) 254:199-208); and the Brassica Bca9 promoter (Lee et al., PlantCell Rep. (2003) 22:268-273).

Certain constructs may also include a gamete tissue preferred promoter,depending upon the various components and the applications in which itis employed. Male gamete preferred promoters include the PG47 promoter,supra as well as ZM13 promoter (Hamilton et al., Plant Mol. Biol. (1998)38:663-669); actin depolymerizing factor promoters (such as Zmabp1,Zmabp2; see for example Lopez et al. Proc. Natl. Acad. Sci. USA (1996)93: 7415-7420); the promoter of the maize petctin methylesterase-likedgene, ZmC5 (Wakeley et al. Plant Mol. Biol. (1998) 37:187-192); theprofilin gene promoter Zmpro1 (Kovar et al., The Plant Cell (2000)12:583-598); the sulphated pentapeptide phytosulphokine gene ZmPSK1(Lorbiecke et al., Journal of Experimental Botany (2005) 56(417):1805-1819); the promoter of the calmodulin binding protein Mpcbp (Reddyet al. J. Biol. Chem. (2000) 275(45):35457-70).

Other components of the vector may be included, also depending uponintended use of the gene. Examples include selectable markers, targetingor regulatory sequences, stabilizing or leader sequences, introns etc.General descriptions and examples of plant expression vectors andreporter genes can be found in Gruber, et al., “Vectors for PlantTransformation” in Method in Plant Molecular Biology and Biotechnology,Glick et al eds; CRC Press pp. 89-119 (1993). The selection of anappropriate expression vector will depend upon the host and the methodof introducing the expression vector into the host. The expressioncassette will also include at the 3′ terminus of the heterologousnucleotide sequence of interest, a transcriptional and translationaltermination region functional in plants. The termination region can benative with the promoter nucleotide sequence of the present invention,can be native with the DNA sequence of interest, or can be derived fromanother source. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al. Mol. Gen.Genet. 262:141-144 (1991); Proudfoot, Cell 64:671-674 (1991); Sanfaconet al. Genes Dev. 5:141-149 (1991); Mogen et al. Plant Cell 2:1261-1272(1990); Munroe et al. Gene 91:151-158 (1990); Ballas et al. NucleicAcids Res. 17:7891-7903 (1989); Joshi et al. Nucleic Acid Res.15:9627-9639 (1987).

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include by way of example, picornavirusleaders, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. Proc. Nat. Acad. Sci. USA 86:6126-6130 (1989);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al.; MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20(1986); human immunoglobulin heavy-chain binding protein (BiP), Macejaket al. Nature 353:90-94 (1991); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. Nature325:622-625 (1987); Tobacco mosaic virus leader (TMV), Gallie et al.(1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al. Virology 81:382-385 (1991). Seealso Della-Cioppa et al. Plant Physiology 84:965-968 (1987). Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast, or to the endoplasmicreticulum, or secreted at the cell's surface or extracellularly, theexpression cassette can further comprise a coding sequence for a transitpeptide. Such transit peptides are well known in the art and include,but are not limited to, the transit peptide for the acyl carrierprotein, the small subunit of RUBISCO, plant EPSP synthase, Zea maysBrittle-1 chloroplast transit peptide (Nelson et al. Plant physiol117(4):1235-1252 (1998); Sullivan et al. Plant Cell 3(12):1337-48;Sullivan et al., Planta (1995) 196(3):477-84; Sullivan et al., J. Biol.Chem. (1992) 267(26):18999-9004) and the like. One skilled in the artwill readily appreciate the many options available in expressing aproduct to a particular organelle. For example, the barley alpha amylasesequence is often used to direct expression to the endoplasmic reticulum(Rogers, J. Biol. Chem. 260:3731-3738 (1985)). Use of transit peptidesis well known (e.g., see U.S. Pat. Nos. 5,717,084; 5,728,925).

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions, such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest. In general, the vectors should befunctional in plant cells. At times, it may be preferable to havevectors that are functional in E. coli (e.g., production of protein forraising antibodies, DNA sequence analysis, construction of inserts,obtaining quantities of nucleic acids). Vectors and procedures forcloning and expression in E. coli are discussed in Sambrook et al.(supra).

The transformation vector comprising the promoter sequence of thepresent invention, or another promoter operably linked to a heterologousnucleotide sequence in an expression cassette and/or the nucleotidesequence of the present invention, can also contain at least oneadditional nucleotide sequence for a gene to be cotransformed into theorganism. Alternatively, the additional sequence(s) can be provided onanother transformation vector.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. Mol.Cell. Biol. 7:725-737 (1987); Goff et al. EMBO J. 9:2517-2522 (1990);Kain et al. BioTechniques 19:650-655 (1995); and Chiu et al. CurrentBiology 6:325-330 (1996).

Selectable reporter genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to,genes encoding resistance to chloramphenicol, Herrera Estrella et al.EMBO J. 2:987-992 (1983); methotrexate, Herrera Estrella et al. Nature303:209-213 (1983); Meijer et al. Plant Mol. Biol. 16:807-820 (1991);hygromycin, Waldron et al. Plant Mol. Biol. 5:103-108 (1985), Zhijian etal. Plant Science 108:219-227 (1995); streptomycin, Jones et al. Mol.Gen. Genet. 210:86-91 (1987); spectinomycin, Bretagne-Sagnard et al.Transgenic Res. 5:131-137 (1996); bleomycin, Hille et al. Plant Mol.Biol. 7:171-176 (1990); sulfonamide, Guerineau et al. Plant Mol. Biol.15:127-136 (1990); bromoxynil, Stalker et al. Science 242:419-423(1988); glyphosate, Shaw et al. Science 233:478-481 (1986); andphosphinothricin, DeBlock et al. EMBO J. 6:2513-2518 (1987).

Scorable or screenable markers may also be employed, where presence ofthe sequence produces a measurable product. Examples include aβ-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (for example, U.S. Pat. Nos.5,268,463 and 5,599,670); chloramphenicol acetyl transferase (Jeffersonet al. The EMBO Journal vol. 6 No. 13 pp. 3901-3907); and alkalinephosphatase. Other screenable markers include the anthocyanin/flavonoidgenes in general (See discussion at Taylor and Briggs, The Plant Cell(1990)2:115-127) including, for example, a R-locus gene, which encodes aproduct that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., in Chromosome Structure andFunction, Kluwer Academic Publishers, Appels and Gustafson eds., pp.263-282 (1988)); the genes which control biosynthesis of flavonoidpigments, such as the maize C1 gene (Kao et al., Plant Cell (1996) 8:1171-1179; Scheffler et al. Mol. Gen. Genet. (1994) 242:40-48) and maizeC2 (Wienand et al., Mol. Gen. Genet. (1986) 203:202-207); the B gene(Chandler et al., Plant Cell (1989) 1:1175-1183), the p1 gene (Grotewoldet al, Proc. Natl. Acad. Sci. USA (1991) 88:4587-4591; Grotewold et al.,Cell (1994) 76:543-553; Sidorenko et al., Plant Mol. Biol.(1999)39:11-19); the bronze locus genes (Ralston et al., Genetics (1988)119:185-197; Nash et al., Plant Cell (1990) 2(11): 1039-1049), amongothers. Yet further examples of suitable markers include the cyanfluorescent protein (CYP) gene (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), the yellowfluorescent protein gene (PhiYFP™ from Evrogen; see Bolte et al. (2004)J. Cell Science 117: 943-54); a lux gene, which encodes a luciferase,the presence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri et al.(1989) EMBO J. 8:343); a green fluorescent protein (GFP) gene (Sheen etal., Plant J. (1995) 8(5):777-84); and DsRed2 where plant cellstransformed with the marker gene are red in color, and thus visuallyselectable (Dietrich et al. (2002) Biotechniques 2(2):286-293).Additional examples include a p-lactamase gene (Sutcliffe, Proc. Nat'l.Acad. Sci. U.S.A. (1978) 75:3737), which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad. Sci.U.S.A. (1983) 80:1101), which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al., Biotech.(1990) 8:241); and a tyrosinase gene (Katz et al., J. Gen. Microbiol.(1983) 129:2703), which encodes an enzyme capable of oxidizing tyrosineto DOPA and dopaquinone, which in turn condenses to form the easilydetectable compound melanin. Clearly, many such markers are available toone skilled in the art.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription or transcript andtranslation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for efficienttransformation/transfection may be employed.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biotechnology, supra; Klein et al,Bio/Technology 10:268 (1992); and Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNA construct may be introduced intothe genomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery, Klein et al., Nature 327: 70-73(1987); electroporation, Fromm et al., Proc. Natl. Acad. Sci. 82: 5824(1985); polyethylene glycol (PEG) precipitation, Paszkowski et al., EMBOJ. 3: 2717-2722 (1984); direct gene transfer WO 85/01856 and EP No. 0275 069; in vitro protoplast transformation, U.S. Pat. No. 4,684,611;and microinjection of plant cell protoplasts or embryogenic callus,Crossway, Mol. Gen. Genetics 202:179-185 (1985). Co-cultivation of planttissue with Agrobacterium tumefaciens is another option, where the DNAconstructs are placed into a binary vector system. See e.g., U.S. Pat.No. 5,591,616; Ishida et al., “High Efficiency Transformation of Maize(Zea mays L.) mediated by Agrobacterium tumefaciens” NatureBiotechnology 14:745-750 (1996). The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al., Science 233: 496-498 (1984),and Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983).

Standard methods for transformation of canola are described at Moloneyet al. “High Efficiency Transformation of Brassica napus usingAgrobacterium Vectors” Plant Cell Reports 8:238-242 (1989). Corntransformation is described by Fromm et al, Bio/Technology 8:833 (1990)and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.See supra and U.S. Pat. No. 5,550,318. Rice transformation is describedby Hiei et al., “Efficient Transformation of Rice (Oryza sativs L.)Mediated by Agrobacterium and Sequence Analysis of the Boundaries of theT-DNA” The Plant Journal 6(2): 271-282 (1994, Christou et al, Trends inBiotechnology 10:239 (1992) and Lee et al, Proc. Nat'l Acad. Sci. USA88:6389 (1991). Wheat can be transformed by techniques similar to thoseused for transforming corn or rice. Sorghum transformation is describedat Casas et al, supra and sorghum by Wan et al, Plant Physicol. 104:37(1994). Soybean transformation is described in a number of publications,including U.S. Pat. No. 5,015,580.

When referring to “introduction” of the nucleotide sequence into aplant, it is meant that this can occur by direct transformation methods,such as Agrobacterium transformation of plant tissue, microprojectilebombardment, electroporation, or any one of many methods known to oneskilled in the art; or, it can occur by crossing a plant having theheterologous nucleotide sequence with another plant so that progeny havethe nucleotide sequence incorporated into their genomes. Such breedingtechniques are well known to one skilled in the art.

The plant breeding methods used herein are well known to one skilled inthe art. For a discussion of plant breeding techniques, see Poehlman(1987) Breeding Field Crops. AVI Publication Co., Westport Conn. Many ofthe plants which would be most preferred in this method are bred throughtechniques that take advantage of the plant's method of pollination.

Backcrossing methods may be used to introduce a gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPlant Breeding Methodology, edit. Neal Jensen, John Wiley & Sons, Inc.(1988). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a plant is obtainedwherein essentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

In certain embodiments of the invention, it is desirable to maintain themale sterile homozygous recessive condition of a male sterile plant,when using a transgenic restoration approach, while decreasing thenumber of plants, plantings and steps needed for maintenance of a plantwith such traits. Homozygosity is a genetic condition existing whenidentical alleles reside at corresponding loci on homologouschromosomes. Heterozygosity is a genetic condition existing whendifferent alleles reside at corresponding loci on homologouschromosomes. Hemizygosity is a genetic condition existing when there isonly one copy of a gene (or set of genes) with no allelic counterpart onthe sister chromosome. In an embodiment, the homozygous recessivecondition results in conferring on the plant a trait of interest, whichcan be any trait desired and which results from the recessive genotype,such as increased drought or cold tolerance, early maturity, changed oilor protein content, or any of a multitude of the many traits of interestto plant breeders. In one embodiment, the homozygous recessive conditionconfers male sterility upon the plant. When the sequence which is thefunctional complement of the homozygous condition is introduced into theplant (that is, a sequence which, when introduced into and expressed inthe plant having the homozygous recessive condition, restores thewild-type condition), fertility is restored by virtue of restoration ofthe wild-type fertile phenotype.

Maintenance of the homozygous recessive condition is achieved byintroducing into a plant restoration transgene construct into a plantthat is linked to a sequence which interferes with the function orformation of male gametes of the plant to create a maintainer or donorplant. The restoring transgene, upon introduction into a plant that ishomozygous recessive for the genetic trait, restores the geneticfunction of that trait, with the plant producing only viable pollencontaining a copy of the recessive allele but not containing therestoration transgene. The transgene is kept in the hemizygous state inthe maintainer plant. By transgene, it is meant any nucleic acidsequence which is introduced into the genome of a cell by geneticengineering techniques. A transgene may be a native DNA sequence, or aheterologous DNA sequence (i.e., “foreign DNA”). The term native DNAsequence refers to a nucleotide sequence which is naturally found in thecell but that may have been modified from its original form. The pollenfrom the maintainer can be used to fertilize plants that are homozygousfor the recessive trait, and the progeny will therefore retain theirhomozygous recessive condition. The maintainer plant containing therestoring transgene construct is propagated by self-fertilization, withthe resulting seed used to produce further plants that are homozygousrecessive plants and contain the restoring transgene construct.

The maintainer plant serves as a pollen donor to the plant having thehomozygous recessive trait. The maintainer is optimally produced from aplant having the homozygous recessive trait and which also hasnucleotide sequences introduced therein which would restore the traitcreated by the homozygous recessive alleles. Further, the restorationsequence is linked to nucleotide sequences which interfere with thefunction or formation of male gametes. The gene can operate to preventformation of male gametes or prevent function of the male gametes by anyof a variety of well-know modalities and is not limited to a particularmethodology. By way of example but not limitation, this can include useof genes which express a product cytotoxic to male gametes (See forexample, U.S. Pat. Nos. 5,792,853; 5,689,049; PCT/EP89/00495); inhibitproduct formation of another gene important to male gamete function orformation (See, U.S. Pat. Nos. 5,859,341; 6,297,426); combine withanother gene product to produce a substance preventing gene formation orfunction (See U.S. Pat. Nos. 6,162,964; 6,013,859; 6,281,348; 6,399,856;6,248,935; 6,750,868; 5,792,853); are antisense to or causeco-suppression of a gene critical to male gamete function or formation(See U.S. Pat. Nos. 6,184,439; 5,728,926; 6,191,343; 5,728,558;5,741,684); interfere with expression through use of hairpin formations(Smith et al. (2000) Nature 407:319-320; WO 99/53050 and WO 98/53083) orthe like. Many nucleotide sequences are known which inhibit pollenformation or function and any sequences which accomplish this functionwill suffice. A discussion of genes which can impact proper developmentor function is included at U.S. Pat. No. 6,399,856 and includes geneswith inhibitory effects such as cytotoxin genes, methylase genes, andgrowth-inhibiting genes. Example of such genes include, but are notlimited to diphtheria toxin A-chain gene (Czako, M. and An, G. (1991)“Expression of DNA coding for Diptheria toxin Chain A is toxic to plantcells” Plant Physiol. 95 687-692. and Greenfield et al PNAS 80:6853(1983), Palmiter et al Cell 50:435 (1987)); cell cycle division mutantssuch as CDC in maize (Colasanti, J., Tyers, M. and Sundaresan, V.,“Isolation and Characterization of cDNA clones encoding a functional P34cdc2 homologue from Zea mays” PNAS 88, 3377-3381 (1991)); the WT gene(Farmer, A. A., Loftus, T. M., Mills, A. A., Sato, K. V., Neill, J.,Yang, M., Tron, T., Trumpower, B. L. and Stanbridge, E. G. Hum. Mol.Genet. 3, 723-728 (1994)); and P68 (Chen, J. J., Pal, J. K., Petryshyn,R., Kuo, I., Yang, J. M., Throop, M. S., Gehrke, L. and London, I. M.“Eukaryotic translation initiation kinases” PNAS 88, 315-319 (1991)).

Further examples of so-called “cytotoxic” genes are discussed supra andcan include, but are not limited to pectate lyase gene pelE, fromErwinia chrysanthermi (Kenn et al J. Bacteroil 168:595 (1986)); T-urf13gene from cms-T maize mitochondrial genomes (Braun et al Plant Cell2:153 (1990); Dewey et al. PNAS 84:5374 (1987)); CytA toxin gene fromBacillus thuringiensis Israeliensis that causes cell membrane disruption(McLean et al J. Bacteriol 169:1017 (1987), U.S. Pat. No. 4,918,006);DNAses, RNAses, (U.S. Pat. No. 5,633,441); proteases, or a genesexpressing anti-sense RNA. A suitable gene may also encode a proteininvolved in inhibiting pistil development, pollen stigma interactions,pollen tube growth or fertilization, or a combination thereof. Inaddition genes that either interfere with the normal accumulation ofstarch in pollen or affect osmotic balance within pollen may also besuitable.

In an illustrative embodiment, the DAM-methylase gene is used, discussedsupra and at U.S. Pat. Nos. 5,792,852 and 5,689,049, the expressionproduct of which catalyzes methylation of adenine residues in the DNA ofthe plant. Methylated adenines will affect cell viability and will befound only in the tissues in which the DAM-methylase gene is expressed.In another embodiment, an α-amylase gene can be used with a maletissue-preferred promoter. During the initial germinating period ofcereal seeds, the aleurone layer cells will synthesize α-amylase, whichparticipates in hydrolyzing starch to form glucose and maltose, so as toprovide the nutrients needed for the growth of the germ (J. C. Rogersand C. Milliman, J. Biol. Chem., 259 (19): 12234-12240, 1984; Rogers, J.C., J. Biol. Chem., 260: 3731-3738, 1985). In an embodiment, theα-amylase gene used can be the Zea mays α-amylase-1 gene. Young et al.“Cloning of an α-amylase cDNA from aleurone tissue of germinating maizeseed” Plant Physiol. 105(2) 759-760 and GenBank accession No. L25805,GI:426481). Sequences encoding α-amylase are not typically found inpollen cells, and when expression is directed to male tissue, the resultis a breakdown of the energy source for the pollen grains, andrepression of pollen development.

One skilled in this area readily appreciates the methods describedherein are applicable to any other crops which have the potential tooutcross. By way of example, but not limitation it can include maize,soybean, sorghum, or any plant with the capacity to outcross.

Ordinarily, to produce more plants having the recessive condition, onemight cross the recessive plant with another recessive plant. This maynot be desirable for some recessive traits and may be impossible forrecessive traits affecting reproductive development. Alternatively, onecould cross the homozygous plant with a second plant having therestoration gene, but this requires further crossing to segregate awaythe restoring gene to once again reach the recessive phenotypic state.Instead, in one process the homozygous recessive condition can bemaintained, while crossing it with the maintainer plant. This method canbe used with any situation in which is it desired to continue therecessive condition. This results in a cost-effective system that isrelatively easy to operate to maintain a population of homozygousrecessive plants.

A sporophytic gene is one which operates independently of the gametes.When the homozygous recessive condition is one which produces malesterility by preventing male sporophyte development, the maintainerplant, of necessity, must contain a functional restoring transgeneconstruct capable of complementing the mutation and rendering thehomozygous recessive plant able to produce viable pollen. Linking thissporophytic restoration gene with a second functional nucleotidesequence which interferes with the function or formation of the malegametes of the plant results in a maintainer plant that produces viablepollen that only contains the recessive allele of the sporophytic geneat its native locus due to the action of the second nucleotide sequencein interfering with pollen formation or function. This viable pollenfraction is non-transgenic with regard to the restoring transgeneconstruct.

In a still further embodiment, a marker gene, as discussed supra, may beprovided in the construct with the restoring transgene. By way ofexample without limitation, use of a herbicide resistant marker, such asbar allows one to eliminate cells not having the restoring transgene. Inyet another example, when using a scorable marker, such as the Ds Red2fluorescent protein, any inadvertent transmission of the transgene canalso be detected visually, and such escapes eliminated from progeny.Clearly, many other variations in the restoring construct are availableto one skilled in the art.

In an illustrative embodiment, a method of maintaining a homozygousrecessive condition of a male sterile plant at a genetic locus isprovided, in which is employed a first nucleotide sequence which is agene critical to male fertility, a second nucleotide sequence whichinhibits the function or formation of viable male gametes, an optionalthird nucleotide sequence which is operably linked to the first sequenceand preferentially expresses the sequence in male plant cells, anoptional fourth nucleotide sequence operably linked to a fourthnucleotide sequence, the fourth sequence directing expression to malegametes, and an optional fifth nucleotide sequence which is a selectableor scorable marker allowing for selection of plant cells.

For example, it is desirable to produce male sterile female plants foruse in the hybrid production process which are sterile as a result ofbeing homozygous for a mutation in the Ms45 gene; a gene which iscritical to male fertility. Such a mutant Ms45 allele is designated asms45 and a plant that is homozygous for ms45 (represented by thenotation ms45/ms45) displays the homozygous recessive male sterilityphenotype and produces no functional pollen. See, U.S. Pat. Nos.5,478,369; 5,850,014; 6,265,640; and 5,824,524. In both the inbred andhybrid production processes, it is maintaining this homozygous recessivecondition is important. When sequences encoding the Ms45 gene areintroduced into a plant that is homozygous recessive for ms45, malefertility results. By the method of the invention, a plant which isms45/ms45 homozygous recessive may have introduced into it a functionalsporophytic Ms45 gene, and thus is male fertile. This gene can be linkedto a gene which operates to render pollen containing the restoringtransgene construct nonfunctional or prevents its formation, or whichproduces a lethal product in pollen, linked to the promoter directingits expression to the male gametes to produce a plant that only producedpollen containing ms45 without the restoring transgene construct.

An example is a construct which includes the Ms45 gene, linked with a5126 promoter, a male tissue-preferred promoter (See U.S. Pat. Nos.5,750,868; 5,837,851; and 5,689,051) and further linked to the cytotoxicDAM methylase gene under control of the polygalacturonase promoter, PG47promoter (See U.S. Pat. Nos. 5,792,853; 5,689,049) in a hemizygoticcondition. Therefore the resulting plant produces pollen, but the onlyviable pollen results from the allele not containing the restoringMs45/DAM methylase construct and thus contains only the ms45 gene. Itcan therefore be used as a pollinator to fertilize the homozygousrecessive plant (ms45/ms45), and progeny produced will continue to bemale sterile as a result of maintaining homozygosity for ms45. Theprogeny will also not contain the introduced restoring transgeneconstruct.

In yet another restoring construct example, the Msca1 gene is linkedwith a Msca1 promoter, and further linked to the Zea mays α-amylase geneunder control of the male tissue-preferred PG47 promoter. The scorablemarker used in an embodiment is DS-RED2.

A desirable result of the process of the invention is that the planthaving the restorer nucleotide sequence may be self-fertilized, that ispollen from the plant transferred to the flower of the same plant toachieve the propagation of restorer plants. (Note that in referring to“self fertilization”, it includes the situation where the plantproducing the pollen is fertilized with that same plant's pollen, andthe situation where two or more identical inbred plants are plantedtogether and pollen from the identical inbred plant pollinate a separatebut identical inbred plant). The restoring transgene construct will notbe present in the pollen cells but it will be contained in 50% of theovules (the female gamete). The seed resulting from theself-fertilization can be planted, and selection made for the seedhaving the restoring transgene construct. The selection process canoccur by any one of many known processes; the most common where therestoration nucleotide sequence is linked to a marker gene. The markercan be scorable or selectable, and allows those plants produced from theseed having the restoration gene to be identified.

In an embodiment of the invention, it is possible to provide that themale gamete-tissue preferred promoter is inducible. Additional controlis thus allowed in the process, where so desired, by providing that theplant having the restoration nucleotide sequences is constitutively malesterile. This type of male sterility is set forth the in U.S. Pat. No.5,859,341. In order for the plant to become fertile, the inducingsubstance must be provided, and the plant will become fertile. Again,when combined with the process of the invention as described supra, theonly pollen produced will not contain the restoration nucleotidesequences.

Further detailed description is provided below by way of instruction andillustration and is not intended to limit the scope of the invention.

EXAMPLE 1

Cloning and Sequencing of the Msca1 Gene

Following mapping of the msca1 mutation to the short arm of chromosome7, a map-based cloning approach was undertaken to clone the msca1 gene.The process of genomic library screenings is commonly known among thoseskilled in the art and is described at Sambrook, J., Fritsch, E. F.,Maniatis T., et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor Lab Press, Plainview, N.Y. (1989).A large mapping population was developed using the msca1-ref allele(West, D P and Albertsen, M C. Three new male-sterile genes 1985. MNL59:87). Markers were used to saturate map this population and aninterval (155 KB) was defined by markers pco144723 and b49 p15.f thatspanned 2 BAC (Bacterial Artificial Chromosome) clones. Sequencing andadditional marker development from the BACs narrowed the interval to 9Kb. Sequence of this region revealed only one open reading frame, codingfor a putative plant-specific glutaredoxin gene. The nucleotide sequenceis shown in FIG. 2 (SEQ ID NO: 1) and the protein sequence is shown inFIG. 3 (SEQ ID NO: 2). Other glutaredoxin family members have previouslybeen shown to have a role in plant development (Shuping Xing, Mario G.Rosso and Sabine Zachgo. ROXY1, a member of the plant glutaredoxinfamily, is required for petal development in Arabidopsis thaliana (2005)Development 132, 1555-1565). Glutaredoxin genes are ubiquitous smallheat-stable oxidoreductases that are believed to function in a widerange of cellular processes from DNA synthesis to protein folding, redoxregulation of transcription and translation, and cellular signaling,among others. In a BLAST comparison of the Msca1 gene, the sequenceswith highest similarity were regions that had 77% identity to aglutaredoxin-like protein.GenBank access No. XP_(—)476652.

Southern analysis of the msca1 reference allele indicated that theglutaredoxin gene was deleted. Sequencing of the reference allelerevealed a 7823 bp deletion, coupled with a 1268 bp insertion 4 Kbdownstream from the glutaredoxin gene. The msca1-ref sequence is shownin FIG. 4 (SEQ ID NO: 3).

EXAMPLE 2

Identification and Cloning of Additional msca1 Alleles

Two additional mutant alleles of msca1 were available, msca1-mg12 andmsca1-6036 (Trimnell M, Fox T, Albertsen M C (2001) New male-sterilemutant allele of Msca1. MNL 75(63):31). Cloning and sequencing of themsca1-mg12 allele revealed a 490 bp deletion in the 3′ region of theglutaredoxin gene. Alignment of a fertile and sterile msca1-mg12 alleleis shown in FIG. 5. Alignment of the glutaredoxin region from awild-type plant (Missouri 17), an msca1-mg12 fertile and sterile plantis shown in FIG. 6. Missing from the sterile plant is the GSH bindingsite, (LPVVFVGGRLLG; SEQ ID NO: 12). A motif for the redox region, CCMC(SEQ ID NO: 11), was present in the gene, and GSH binding region,LPVVFVGGRLLG (SEQ ID NO: 12), present in fertile plant, is absent in thesterile mutant as can be seen in the alignment shown in FIG. 6.

In cloning and sequencing of the rascal-6036 allele, a ˜850 bp insertionwas detected. Alignment showing comparison with a fertile plant versusthe sterile allele is shown in FIG. 7. This insertion created an 8 bphost site duplication (GTCGAGAA) and it also appears to contain smallperfect TIRs (See the region of the sequence following base 854 in FIG.7, marked at the start with “TIR”). It was also noted there is ˜200 bpof significant homology at the ends of the insertion, reminiscent of aplant transposon. A graphic alignment of the msca1 coding region,genomic region, the reference allele, the msca1-mg12 allele andrascal-6036 allele is shown in FIG. 8.

EXAMPLE 3

Identification of Promoter

Upstream of the likely translational start codon at 1133 bp of SEQ IDNO: 1 of Msca1, 1132 bp of DNA was present in the genomic clone ofMsca1. A reasonable TATA box was observed by inspection, starting atbase 921 of SEQ ID NO: 1 and about 200 bp upstream of the translationalstart codon. See FIG. 9, which is SEQ. ID NO 15. The putative TATA box(TATAAAA) is underlined. Thus, the present invention encompasses a DNAmolecule having a nucleotide sequence of SEQ ID NO: SEQ ID NO: 15 (FIG.9), or those with sequence identity, which hybridize to same understringent conditions and fragments, and having the function of a maletissue-preferred regulatory region.

EXAMPLE 4

Library Screening to Identify Msca1 from Rice

As noted above, Msca1 is a male fertility gene in maize. When it ismutated, and made homozygous recessive, male sterility will result. Anorthologue of Msca1 was identified in rice. The rice Deleteagenepopulation was prepared and used to screen for individuals harboringdeletions of the msca1 gene. (Xin Li et. al A fast neutron deletionmutagenesis-based reverse genetics system for plants. The Plant JournalVolume 27 Page 235-August 2001). With this process, random deletionlibraries are produced using fast neutrons to cause mutations. Thelibraries are screened for specific deletion mutants using polymerasechain reaction (PCR). In a typical protocol, 18 seeds from lines arepooled, planted, seedlings collected and genomic DNA isolated from thetissue. The DNA so isolated from all the mutated lines is collected intopools, beginning with mega pools, each having DNA of 2592 lines. A pairof primers are selected that are specific to sequences which flanks agene targeted for deletion along with another pair of internally nestedprimers. The primers used for msca1 were as follows:

(SEQ ID NO: 20) 5′ TGAGCATGCATGCTAAGCTAGTACTCCAGC (SEQ ID NO: 21) 5′GTGATCCTCTCTGATGGTGACAACGAAGAC

The goal is to screen the library with one primer specific to the geneand a primer specific to the insertion element in such a way that onecan discriminate between amplification of wild-type DNA from insertionDNA in large pools. The primers amplify both wild-type and mutant genes,but the PCR extension time is reduced in order to suppress amplificationof the wild-type DNA. A long extension time is first used to confirmprimer quality, then a shorter extension time to determine under whatconditions amplification of wild-type DNA is suppressed. This time isused to screen the mega pools. A second round of PCR using nestedprimers is used to increase sensitivity. Gel electrophoresis detects thepresence of amplified fragments in deletion alleles, and if a band isfound in a mega pool, PCR analysis continues on smaller pool groupsuntil a single plant is identified.

Primers derived from the rice msca1 gene yielded a putative deletionproduct in the initial screen of the 10 mega pools (having 2592 familiesper pool), which encompasses the entire mutant population. From this adeletion product in mega pool 7 was identified. This product was clonedand sequenced and was identified as a deletion allele of the rice msca1gene as shown in FIG. 13. Subsequent screenings were performed on thenine superpools comprised of 288 families per pool, the 16 pools (having16 families per pool), the 9 sub pools (two families per pool) toultimately identify the individual families harboring the deletion. Twofamilies were identified. Seed from each of these families were grownand genotyped using a set of wild-type primers and a set of primers thatspecifically amplify across the deletion. Family 21-77 was identified ascontaining the deletion in the rice msca1 gene and 2 plants within thisfamily were homozygous for the mutation. Plants from family 21-77 weregrown to maturity and male fertility phenotype was noted. The two plantsgenotyped as being homozygous for the msca1 deletion were completelymale sterile, whereas sibling plants were male fertile, confirming thefunction of Msca1 in rice as being required for male fertility,analogous to the maize Msca1 function. Cytological examination of theanthers showed them to be small and mis-shapened with no evidence ofmicrospore development. Stigmas from mutant flowers appeared to benormal. Crosses onto one of the mutant panicles resulted in seed set,demonstrating the female flower is viable. The second mutant had itspanicle bagged and did not set any seed, confirming the male sterilityphenotype.

EXAMPLE 5

Cloning and Sequence of Msca1 from Rice

A wildtype rice Msca1 gene from plant variety M202 (Johnson, C. W.,Carnahan, H. L., Tseng, S. T., Oster, J. J., and Hill, J. E.Registration of “M202” rice. Crop Science, Vol 26. January-February.1986 page 198) was cloned and sequenced using methods described supraand the 2860 base pairs of nucleotide sequence is shown in FIG. 10 (SEQID NO: 16). The putative amino acid sequence is shown in FIG. 11 (SEQ IDNO: 17). A motif for the redox region, CCMC, and the GSH binding regionVPVVFVGGRLLG (SEQ ID NO: 11 and 25, respectively) is present in the ricemsca1 protein as shown in FIG. 11. The subclone of this gene is verysimilar in size and sequence composition to the maize Msca1 genomicclone that has been shown to complement the maize msca1 mutation.

EXAMPLE 6

Identification of Msca1 Promoter from Rice

Upstream of the likely translational start codon at 1317 bp of SEQ IDNO: 16 (FIG. 10) of Msca1, 1316 bp of DNA was present in the genomicclone of rice Msca1. A reasonable TATA box was observed by inspection,starting at base 1008 of SEQ ID NO: 16 and about 200 bp upstream of thetranslational start codon. See FIG. 12, which is SEQ. ID NO: 18 showingthe promoter. The putative TATA box (TATATATATATA) (SEQ ID NO: 22). isunderlined. Thus, the present invention encompasses a DNA moleculehaving a nucleotide sequence of SEQ ID NO: 18 (or those with sequenceidentity or which hybridize under stringent conditions or fragments ofsame) and having the function of a male tissue-preferred regulatoryregion.

EXAMPLE 7

Construct Preparation with Msca1 Gene

A construct designated PHP27077 is made by assembling following DNAcomponents:

-   1. The plasmid pSB11 backbone DNA (pSB31 lacking the EcoRI fragment    carrying the 35SGUS and 35SBAR genes, Ishida et al., Nature    Biotechnol. (1996) 14:745-750). This DNA backbone contains T-DNA    border sequences and the replication origin from pBR322.-   2. The 35S:PAT gene which encodes the enzyme phosphinothricin    acetyltransferase (PAT) from Streptomyces viridochomagenes    (nucleotides 6-557 from accession number A02774, Strauch et al.    1988, EP 0275957-A) under the transcriptional control of the    cauliflower mosaic virus (CaMV) 35S promoter and terminator    (nucleotides 6906-7439, and 7439-7632, respectively from Franck et    al. 1980, Cell 21: 285-294).-   3. The Msca1 sequence as set forth in FIG. 2 (SEQ ID NO: 1)

EXAMPLE 8

Transformation of Maize msca1 Plants

A male-sterile female which was homozygous for the msca1-ref mutantallele, (msca1) was repeatedly crossed with bulked pollen from maizeHi-type II plants (Armstrong 1994, In: Freeling and Walbot (eds). TheMaize Handbook. Springer, N.Y., pp 663-671) resulting in theintrogression of this msca1 allele in transformation amenable maizegermplasm over multiple generations. The resultant source of materialfor transformation consisted of embryos segregating (1:1 or 3:1) formsca1 and allowed for both transformation directly into a homozygousmsca1 background and to test the genetic complementation of the msca1mutation in T₀ plants. Agrobacterum-mediated transformation wasperformed according to Zhao et al. 1999, (U.S. Pat. No. 5,981,840).Genotyping and molecular analysis (integration and plant transcriptionunits/PTU) of transformants were done according Cigan et al., (Sex.Plant. Reprod. 1 (2001) 4:135-142). Single copy, intact PTU events wereidentified by Southern analysis. Msca1 genotyping was accomplished byPCR of the single copy events. No morphological difference was observedbetween the transgenic plants and the non-transgenic control plantsexcept for the degree of male fertility. Transformants were completelymale fertile while non-transgenic control plants were completely malesterile, indicating that the expression of the Msca1 gene complementedthe homozygous recessive msca1 male sterile phenotype.

EXAMPLE 9

Transformation of Msca1 Plants with a Construct with Msca1 Having aFrameshift

A second construct, PHP27618, is essentially the same as PHP27077, buthas had a frameshift introduced into the msca1 gene, adding four basepairs at position 1508 of the sequence SEQ ID NO: 1_) to disrupt theputative translation of the gene. Single copy, intact PTU events wereidentified by Southern analysis. Msca1 genotyping was done by PCR on thesingle copy events. Male fertility/sterility phenotype scores were takenat flowering. Results show that the frameshifted Msca1 genomic fragmentdoes not restore male fertility to msca1/msca1 plants which indicatesthat the putative translation product shown in FIG. 3 is the correcttranslational frame for the Msca1 gene. Specific results are shownbelow.

Events Genotype Fertility PHP27077 - 10 single copy events 2 Msca1/Msca1F 1 Msca1/msca1 F 7 msca1/msca1 F PHP27618 (frame shift) - 6 single copyevents 2 Msca1/msca1 F 4 msca1/msca1 S

EXAMPLE 10

Expression of Promoter

A construct PHP28154 was prepared with bases 1-1109 of the Msca1promoter (SEQ ID NO: 15) and also included the cyan fluorescent protein(CFP) marker (Bolte et al. (2004) J. Cell Science 117: 943-54 and Katoet al. (2002) Plant Physiol 129: 913-42). Following Agrobacteriumtransformation (into GS3, the events were subjected to quantitativepolymerase chain reaction (QPCR) of the PAT gene to determine copynumber. Duplicate plants for most of the events were sent to thegreenhouse. Tissue dissection from the duplicates was initiated ataround the three to four leaf stage through the eight leaf stage. Nosignal could be seen in vegetative meristems, or in any other plant parte.g. roots, leaves. Well after the meristem had transitioned into afloral structure and anthers were being formed, CFP signal could beobserved in the anther initials up through a developed anther (˜1 mm).The signal disappeared once the anther had fully developed pollen mothercells, just prior to meiosis. This observation of CFP expressiondemonstrates the role of Msca1 in determining anther morphology.

EXAMPLE 11

Complementation Study of an Alternative Msca1 Promoter Fragment

Another construct, PHP27612, was prepared which included a smallerportion of the Msca1 genomic sequence. This fragment included 1291 basesof the genomic Msca1, starting from position 610 to 1900 bp SEQ ID NO: 1(FIG. 2) which corresponds to a promoter length of 522 bases, from 610to 1132 bases of the promoter sequence in FIG. 9. (SEQ ID NO: 15). Theconstruct was prepared with the following components:

-   1. The plasmid pSB11 backbone DNA (pSB31 lacking the EcoRI fragment    carrying the 35SGUS and 35SBAR genes, Ishida et al., Nature    Biotechnol. (1996) 14:745-750). This DNA backbone contains T-DNA    border sequences and the replication origin from pBR322.-   2. The PG47PRO:ZM-AA1 gene which contains alpha-amylase 1 coding    region from Zea mays.-   3. LTP2:DS-RED2 (ALT1) which contains red florescence coding region    (a variant of Discosoma sp. red fluorescent protein (DsRed), from    Clontech mutated to remove BstEII site, codon sequence unchanged)    driven by LTP2 promoter, supra.

Plants were transformed with PHP27612 as described supra, into msca1sterile mutants. The introduction of the construct did not complementthe mutation and the plants remained sterile, indicating that there areregulatory elements outside of this 1291 basepair fragment that arerequired for normal Msca1 biological function.

As is evident from the above, nucleotide sequences which map to theshort arm of chromosome 7 of the Zea mays genome, at the same site asthe Msca1 gene, and its alleles, are genes critical to male fertility inplants, that is, are necessary for fertility of a plant, or, whenmutated from the sequence found in a fertile plant, cause sterility inthe plant. Thus it can be seen that the invention achieves at least allof its objectives.

What is claimed is:
 1. A method of maintaining a homozygous recessivecondition of a male sterile plant, the method comprising: (a) providinga first plant comprising homozygous recessive alleles of a Msca1 geneand wherein said plant is male sterile; (b) providing a second plantcomprising homozygous recessive alleles of Msca1 gene and a construct inthe hemizygous condition, the construct comprising: (i) a firstnucleotide sequence comprising the Msca1 nucleotide sequence, that whenexpressed in the first plant would restore male fertility; (ii) a secondnucleotide sequence that when expressed inhibits the function orformation of viable male gametes in the second plant, such that viablemale gametes are produced in the second plant containing the recessivealleles of Msca1 and that do not contain the construct; (iii) a thirdnucleotide sequence regulating expression of the first nucleotidesequence, the third nucleotide sequence selected from the groupconsisting of: a) SEQ ID NO: 18; and b) a nucleic acid sequence isolatedfrom the 5′ region natively associated with the Msca1 coding region inrice, wherein the sequence confers male tissue preferred expression; (c)fertilizing the first plant with the male gametes of the second plant toproduce progeny which maintain the homozygous recessive condition of thefirst plant.
 2. The method of claim 1, wherein the second nucleotidesequence is selected from the group consisting of the nucleotidesequence of the DAM methylase gene, Zea mays alpha amylase gene, and acytotoxin encoding gene.
 3. The method of claim 1, wherein the secondnucleotide sequence is operably linked to a fourth nucleotide sequence,the fourth nucleotide sequence directing expression preferentially tomale gametes.
 4. The method of claim 3, wherein the fourth nucleotidesequence is selected from the group consisting of the regulatory regionof the polygalacturonase 47 gene', Zm13 , pectin methylesterase gene,calmodulin binding protein gene, actin depolymerizing factor gene,profilin gene, and sulphated pentapeptide phytosulphokine gene.
 5. Themethod of claim 1, wherein said construct further comprises a fifthnucleotide sequence encoding a product, the expression of which iscapable of being used for selection of plant cells having the construct.6. The method of claim 5 wherein the fifth nucleotide sequence isselected from the group consisting of red fluorescent gene, cyanfluorescent protein gene, yellow fluorescent protein gene, luciferasegene, green fluorescent protein gene, anthocyanin p1 gene andphosphinothricin acetyltransferase encoding gene.
 7. The method of claim5, further comprising selecting for said second plant by identifyingplants having said construct.
 8. A method of producing seed from a planthaving male gametes, the method comprising: (a) introducing into a malesterile plant comprising homozygous recessive alleles of a Msca1 gene, aconstruct in the hemizygous condition comprising: (i) a first nucleotidesequence comprising the Msca1 nucleotide sequence that when expressedrestores male fertility; (ii) a second nucleotide sequence, that whenexpressed inhibits the function or formation of male gametes in theplant, such that the plant produces viable male gametes that do notcontain the construct; (iii) a third nucleic acid sequence operablylinked to the first nucleic acid sequence, the third nucleic acidsequence selected from the group consisting of: a) SEQ ID NO: 18; and b)a nucleic acid sequence isolated from the 5′ region natively associatedwith the Msca1 coding region in rice, wherein the sequence confers maletissue preferred expression; (b) self fertilizing the plant; and (c)producing the seed which contain the construct.
 9. The method of claim8, wherein the second nucleotide sequence is operably linked to a fourthnucleotide sequence, the fourth nucleotide sequence directing expressionpreferentially to male gametes.
 10. The method of claim 8, saidconstruct further comprising a fifth nucleotide sequence encoding aproduct capable of being used for selection of plant cells containingthe construct.
 11. The method of claim 10, further comprisingidentifying plants having said construct.
 12. A method of restoring malefertility in a male sterile plant, the male sterile plant comprisinghomozygous recessive alleles of a gene that impacts male fertility, themethod comprising introducing into said plant a first nucleotidesequence that is the functional complement of the homozygous recessivecondition, said first nucleotide sequence operably linked to a secondnucleic acid molecule, said second nucleotide sequence selected from thegroup consisting of: (a) SEQ ID NO: 18; and (b) a nucleic acid sequenceisolated from the 5′ region natively associated with the Msca1 codingregion in rice, wherein the sequence confers male tissue preferredexpression.